Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 184-198).

TRANSCRIPTIONAL ANALYSIS OF HYDROGENASE GENES IN RHODOBACTER SPHAEROIDES O.U.001 In photosynthetic non-sulphur bacteria, hydrogen production is catalyzed by nitrogenases and hydrogenases. Hydrogenases are metalloenzymes that are basically classified into: the Fe hydrogenases, the Ni-Fe hydrogenases and metal-free hydrogenases. Two distinct Ni-Fe hydrogenases are described as uptake hydrogenases and bidirectional hydrogenases. The uptake hydrogenases are membrane bound dimeric enzymes consisting of small (hupS) and large (hupL) subunits, and are involved in uptake and the recycling of hydrogen, providing energy for nitrogen fixation and other metabolic processes. In this study the presence of the uptake hydrogenase genes was shown in Rhodobacter sphaeroides O.U.001 strain for the first time and hupS gene sequence was determined. The sequence shows 93% of homology with the uptake hydrogenase hupS of R.sphaeroides R.V. There was no significant change in growth of the bacteria at different concentrations of metal ions (nickel, molybdenum and iron in growth media). The effect of metal ions on hydrogen production of the organism was also studied. The maximum hydrogen gas production was achieved in 8.4&micro / M of nickel and 0.1 mM of iron containing media. The expression of uptake hydrogenase genes were examined by RT-PCR. Increasing the concentration of Ni++ up to 8.4&micro / M increased the expression of uptake hydrogenase genes (hupS). At varied concentrations of Fe-citrate (0.01 mM-0.1 mM) expression of hupS was not detected until hydrogen production stopped. These results will be significant for the improvement strategies of Rhodobacter sphaeroides O.U.001 to increase hydrogen production efficiency. In order to examine the presence of hupL genes, different primers were designed. However, the products could not be observed by PCR.

Universiẗat, Diss., 2003--München.

Bacteria swim towards improving conditions by controlling flagellar activity via signals (CheY) sent from chemosensory protein clusters, which respond to changing stimuli. The best studied chemotactic bacterium, E. coli, has one transmembrane chemosensory protein cluster controlling flagellar behaviour. R. sphaeroides has two clusters, one transmembrane and one cytoplasmic. The roles of the two clusters in regulating swimming and chemosensory behaviour are explored here. Newly-developed software was used to measure the effect of deleting or mutating each chemotaxis protein on unstimulated swimming and on the chemosensory response to dynamic change. New behaviours were identified by using much larger sample sizes than previous studies. R. sphaeroides chemotaxis mutants were classified as (i) stoppy unresponsive; (ii) smooth unresponsive or (iii) stoppy inhibited compared to wildtype swimming and chemosensory behaviour. The data showed that the ability to stop during free-swimming is not necessarily connected to the ability to respond to a chemotaxis challenge. The data suggested a new model of connectivity between the two chemosensory pathways. CheY₃ and CheY₄ are phosphorylated by the transmembrane polar cluster in response to external chemoeffector concentrations. CheY₆-P produced by the cytoplasmic cluster is a requirement for chemotaxis, whether or not the polar cluster is able to produce CheY₆-P. CheY₆-P stops the motor, whereas CheY_3,4-P allow smooth swimming. When chemoeffector levels fall, the signals through CheY_3,4 fall, allowing CheY₆-P to bind and stop the motor. As the polar cluster adapts to the fall by the action of the adaptation proteins CheB₁ and CheR₂, the concentration of CheY_3,4-P increases again, to compete with CheY₆-P and allow periods of smooth swimming. Under aerobic conditions, the cytoplasmic cluster controls the basal stopping frequency and does not appear to respond to external chemoeffector changes. The role of the adaptation proteins in resetting the signalling state in R. sphaeroides is unclear, particularly the roles of the proteins associated with the cytoplasmic cluster, CheB₂ and CheR₃. Tandem mass spectrometry was used to identify glutamate and glutamine (EQ) sites on the cytoplasmic R. sphaeroides chemoreceptor TlpT that are deamidated and methylated by the R. sphaeroides adaptation homologues. In E. coli, adaptation sites are usually EQ/EQ pairs. However the sites reported in TlpT vary at the first residue in the pair. Mutation of the putative EQ adaptation sites caused changes in adaptation, suggesting that CheY₆-P levels are controlled and reset by CheB₂ and CheR₃ controlling the adaptation state of TlpT.

Durch die vorliegende Arbeit wurde gezeigt, dass Rhodobacter sphaeroides das Potenzial besitzt, umweltverträglich photoheterotroph Wasserstoff als alternativer, erneuerbarer Energieträger zu erzeugen. Aus genomischen und transkriptomischen Erkenntnissen konnten Rückschlüsse auf Ansatzpunkte für weitere Optimierungen getroffen werden. Durch ein neues Minimalmedium, welches zukünftig sogar einen Beitrag zur Abfallbeseitigung leisten kann, wurde ein wichtiger Schritt hinsichtlich der industriellen Anwendbarkeit von R. sphaeroides für die biologische Wasserstoffproduktion gemacht.:Danksagung Datenverfügbarkeit Inhaltsverzeichnis Abbildungsverzeichnis Tabellenverzeichnis Abkürzungsverzeichnis 1. Einleitung 1.1 Wasserstoff 1.1.1 Wasserstoff als Energieträger 1.1.2 Herstellung von Wasserstoff 1.1.2.1 Konventionelle Wasserstoffproduktion 1.1.2.2 Biologische Wasserstoffproduktion 1.1.2.3 Biologische Wasserstoffproduktion aus Abfällen 1.2 Photosynthetische Bakterien 1.2.1 Rhodobacter sphaeroides im Kontext der biologischen Wasserstoffproduktion 1.2.2 An der Wasserstoffproduktion beteiligte Enzyme 1.3 Third Generation-Sequencing Technologien 2. Zielstellung 3. Material 3.1 Chemikalien 3.2 Medien und Pufferlösungen 3.2.1 Van Niel´s Yeast Medium 3.2.2 Medium nach Krujatz et al. (2014) 3.2.3 RÄ-Medium nach Mougiakos et al. (2019) 3.2.4 PY (Peptone Yeast) Agarmedium 3.2.5 2x YT Medium 3.2.6 LB Medium 3.2.7 GYCC Medium 3.2.8 SOB Medium 3.2.9 SOC Medium 3.2.10 Pufferlösungen 3.3 Mikroorganismen 3.4 Molekularbiologische Reagenzien und Primer 3.5 Plasmide 3.5.1 pCas9 3.5.2 pRKPOL2 3.5.3 pSUPPOL2Sca 3.5.4 pBBRBB-Ppuf843-1200-DsRed 3.5.5 pBBR_cas9_NT 3.6 Geräte 4. Methoden 4.1 Rhodobacter sphaeroides Dauerkultur in Van Niel´s Yeast Medium 112 (ohne Wasserstoffproduktion) 4.2 Rhodobacter sphaeroides Batch-Kultivierung 4.2.1 Kultivierung in Medium nach Krujatz et al. (2014); Vollmedium mit Wasserstoffproduktion 4.2.2 Kultivierung in Fruchtsaftmedium 4.3 Rhodobacter sphaeroides Kultivierung mit kontinuierlicher Aufzeichnung von Temperatur, pH, optischer Dichte, Wasserstoffproduktion und Gasanalyse 4.4 Zellernte 4.5 Nukleinsäureextraktion mit dem MasterPureTM Complete RNA and DNA Purification Kit 4.6 DNase-Abbau 4.7 RNase-Abbau 4.8 Qualitätskontrolle der RNA und DNA mit dem Agilent 2100 Bioanalyzer 4.9 Reverse Transkription und Probenaufreinigung 4.10 qRT-Polymerasekettenreaktion 4.11 Etablierung der CRISPR-Cas9- Methodik bei Rhodobacter sphaeroides – Gen-Knockout der Hydrogenase Untereinheit hupL mit CRISPR-Cas9 4.11.1 Anzucht der Escherichia coli Stämme mit und ohne Plasmid 4.11.2 Plasmid Extraktion mit GeneJET Plasmid Miniprep Kit (#K0502, Thermo Scientific) 4.11.3 Restriktionsverdau zur Vektorlinearisierung 4.11.4 Design der guideRNA 4.11.5 Phosphorylierung der guideRNA 4.11.6 Ligation der guideRNA in pCas9 4.11.7 Transformation pCas9_hupL1/hupL2 in Escherichia coli JM109 durch chemische Kompetenz 4.11.8 Colony-PCR zum Insertnachweis hupL1&2 in pCas9 mit GoTaq® G2 Green Master Mix (Promega) 4.11.9 Konstruktion weiterer Vektoren mit CRISPR-Cas9 Maschinerie aus pCas9_hupL1/2 4.12 Genomeditierung in Rhodobacter sphaeroides 4.12.1 Transformation durch chemische Kompetenz mit PEG-Methode 4.12.2 Transformation durch chemische Kompetenz nach Hanahan et al. (1991) 4.12.3 Konjugation mit Escherichia coli S17-1 4.12.4 Elektroporation 4.12.5 Bioballistische Genomeditierung mit PDS-1000/He Particle Delivers System (BIORAD) 4.12.6 Konjugation mit Escherichia coli S17-1 nach Mougiakos et al. (2019) 65 4.13 Probenvorbereitung für Sequenzierungen 4.13.1 Illumina MiSeq (Genomsequenzierung) 4.13.2 MinION (Genomsequenzierung) 4.13.3 Illumina HiSeq (Transkriptomsequenzierung) 4.14 Bioinformatische Methoden 4.14.1 Genomsequenzierung (Re-Sequenzierung) 4.14.2 Transkriptom-Datenanalyse 5. Ergebnisse und Diskussion 5.1 Schrittweise Reduktion des Vollmediums nach Krujatz et al. (2014) zum Fruchtsaft-Minimalmedium 5.2 Untersuchung der Wasserstoffproduktion in Fruchtsaft-Minimalmedium 5.3 Kontinuierliche Aufzeichnung von Prozessdaten im 1,2 L Bioreaktor 5.3.1 Vergleich der Reaktorläufe in Vollmedium nach Krujatz et al. (2014), Trauben- und Ananas-Minimalmedium der Stämme DSM 158 und SubH2 5.3.2 Prozessgasanalyse 5.4 Analyse des Genoms 5.4.1 Multiples Sequenzalignment der kompletten genomischen Assemblies von Rhodobacter sphaeroides 5.4.2 MiSeq-Sequenzierung des Stammes Rhodobacter sphaeroides 2.4.1. SubH2 5.4.2.1 Bioinformatische Funktionsanalyse von SNPs 5.4.2.2 SNP-Analyse mittels Homology-Modeling 5.4.3 Genomische Architekturanalyse mittels MinION Sequenzierung der Rhodobacter sphaeroides Stämme DSM 158 und 2.4.1. SubH2 5.4.4 Vergleich der MiSeq- und MinION Genomanalysen 5.5 Analyse des Transkriptoms 5.6 Analyse der Genexpression mit qRT-PCR im Vergleich mit der Wasserstoffproduktion 5.7 CRISPR-Cas9 zum Plasmid-basierten hupL Knock-out 5.7.1 Erstellung der Plasmide pCas9_hupL1 und pCas9_hupL2 5.7.2 PEG-basierte Transformation nach Fornari et al. (1982) 5.7.3 Transformation mittels Elektroporation 5.7.4 Erstellung weiterer Vektoren mit CRISPR-Cas9_Maschinerie aus pCas9_hupL1&2 5.7.5 Transformation mittels Konjugation I 5.7.6 Bioballistische Transformation 5.7.7 Problembehandlung zur Transformation 5.7.8 Transformation mittels Konjugation II 6 Zusammenfassung 7 Ausblick 8 Summary Literaturverzeichnis Anhangsverzeichnis Anhang Versicherung

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Perceiving and responding to the environment is key to survival. Using the prokaryotic equivalent of a nervous system – the chemotaxis system – bacteria sense chemical stimuli and respond by adjusting their movement accordingly. In chemotactic bacteria, such as the well-studied E. coli, environmental nutrient sensing is achieved through a membrane embedded protein array that specifically clusters at the cell poles. Signalling to the motor is performed by activation of the CheA kinase, which phosphorylates CheY and CheB. CheY-P tunes the activity of the flagellar motor while CheB-P, together with CheR is involved in adaptation to the stimulus. In E. coli, a dedicated phosphatase terminates the signal. Most bacterial species however, have a much more complex chemotaxis network. Rhodobacter sphaeroides, a model organism for complex chemotaxis systems, has one membrane-embedded chemosensory array and one cytoplasmic chemosensory array, plus several homologs of the E. coli chemotaxis proteins. Signals from both arrays are integrated to control the rotation of a single start-stop flagellar motor. The phosphorelay network has been studied extensively through in vitro phosphotransfer while in vivo studies have established the components of each array and the requirements for formation. Mathematical modelling has also contributed towards inferring connectivities within the signalling network. Starting by constructing a two-hybrid-based interaction network focused on the components of the cytoplasmic chemosensory array, this thesis further addresses its associated adaptation network through a series of in vivo techniques. The swimming behaviour of series of deletion mutants involving the adaptation network of R. sphaeroides is characterised under steady state conditions as well as upon chemotactic stimulation. New connectivities within the R. sphaeroides chemotaxis network are inferred from analysing these data together with results from in vivo photoactivation localisation microscopy of CheB₂. The experimental results are used to propose a new model for chemotaxis in R. sphaeroides.

Harvesting solar energy can potentially be a promising solution to the energy crisis now and in the future. However, material and processing costs continue to be the most important limitations for the commercial devices. A key solution to these problems might lie within the development of bio-hybrid solar cells that seeks to mimic photosynthesis to harvest solar energy and to take advantage of the low material costs, negative carbon footprint, and material abundance. The bio-photoelectrochemical cell technologies exploit biomimetic means of energy conversion by utilizing plant-derived photosystems which can be inexpensive and ultimately the most sustainable alternative. Plants and photosynthetic bacteria harvest light, through special proteins called reaction centers (RCs), with high efficiency and convert it into electrochemical energy. In theory, photosynthetic RCs can be used in a device to harvest solar energy and generate 1.1 V open circuit voltage and ~1 mA cm-2 short circuit photocurrent. Considering the nearly perfect quantum yield of photo-induced charge separation, efficiency of a protein-based solar cell might exceed 20%. In practice, the efficiency of fabricated devices has been limited mainly due to the challenges in the electron transfer between the protein complex and the device electrodes as well as limited light absorption. The overarching goal of this work is to increase the power conversion efficiency in protein-based solar cells by addressing those issues (i.e. electron transfer and light absorption). This work presents several approaches to increase the charge transfer rate between the photosynthetic RC and underlying electrode as well as increasing the light absorption to eventually enhance the external quantum efficiency (EQE) of bio-hybrid solar cells. The first approach is to decrease the electron transfer distance between one of the redox active sites in the RC and the underlying electrode by direct attachment of the of protein complex onto Au electrodes via surface exposed cysteine residues. This resulted in photocurrent densities as large as ~600 nA cm-2 while still the incident photon to generated electron quantum efficiency was as low as %3 × 10-4. 2- The second approach is to immobilize wild type RCs of Rhodobacter sphaeroides on the surface of a Au underlying electrode using self-assembled monolayers of carboxylic acid terminated oligomers and cytochrome c charge mediating layers, with a preferential orientation from the primary electron donor site. This approach resulted in EQE of up to 0.06%, which showed 200 times efficiency improvement comparing to the first approach. In the third approach, instead of isolated protein complexes, RCs plus light harvesting (LH) complexes were employed for a better photon absorption. Direct attachment of RC-LH1 complexes on Au working electrodes, resulted in 0.21% EQE which showed 3.5 times efficiency improvement over the second approach (700 times higher than the first approach). The main impact of this work is the harnessing of biological RCs for efficient energy harvesting in man-made structures. Specifically, the results in this work will advance the application of RCs in devices for energy harvesting and will enable a better understanding of bio and nanomaterial interfaces, thereby advancing the application of biological materials in electronic devices. At the end, this work offers general guidelines that can serve to improve the performance of bio-hybrid solar cells.